Figure 2.

DNA interstrand crosslinking assay and in vitro activity of EC2629. (a) Calf thymus DNA mixed with varying concentrations of EC2629 with (filled circle) or without (open circle) 0.1 mM DTT were incubated at 37 °C for 2 h. Ethidium bromide was then mixed with above solution, incubated at room temperature for 2 h and fluorescence (Ex: 535 nm, Em: 605 nm) measured. The DNA solution was then heat denatured, cooled and fluorescence re-measured. The ratio of the fluorescence after heating to the fluorescence before heating yields the % DNA cross-linking. (b) KB cells were pulsed for 2 h with increasing concentrations of EC2629 in the absence (filled circle) or presence of 100 µM folic acid (open circle) as a competitor. Points, average of three wells; bars, s.d.